Ecotoxicology and Environmental Safety 162 (2018) 129–138

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Mining alfalfa (Medicago sativa L.) nodules for salinity tolerant non-rhizobial T
bacteria to improve growth of alfalfa under salinity stress
⁎ ⁎
Fatemeh Nooria, Hassan Etesamib, , Hamid Najafi Zarinia, , Nayer Azam Khoshkholgh-Simac,
Ghasem Hosseini Salekdehc, Farshad Alishahib
a
Department of Biotechnology and Plant Breeding, Sari Agricultural Sciences and Natural Resources University, Sari, Iran
b
Agriculture & Natural resources Campus, Faculty of Agricultural Engineering & Technology, Department of Soil Science, University of Tehran, Tehran 31587-77871, Iran
c
Agriculture Biotechnology Research Institute of Iran (ABRII), Agriculture Research, Education and Extension Organization (AREEO), Karaj, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: There are fewer reports on plant growth promoting (PGP) bacteria living in nodules as helper to tolerance to
Co-inoculation abiotic stress such as salinity and drought. The study was conducted to isolate rhizobial and non-rhizobial
Nodule salinity and drought tolerant bacteria drought and salinity tolerant bacteria from the surface sterilized root nodules of alfalfa, grown in saline soils, and
Rhizobia evaluate the effects of effective isolates on plant growth under salt stress. Based on drought and salinity tolerance
Klebsiella sp.
of bacterial isolates and having multiple PGP traits, two non-rhizobial endophytic isolates and one rhizobial
Kosakonia cowanii
Legume
endophytic isolate were selected for further identification and characterization. Based on partial sequences of
16 S rRNA genes, non-rhizobial isolates and rhizobial isolate were closely related to Klebsiella sp., Kosakonia
cowanii, and Sinorhizobium meliloti, respectively. None of the two non-rhizobial strains were able to form nodules
on alfalfa roots under greenhouse and in vitro conditions. Co-inoculation of alfalfa plant with Klebsiella sp. A36,
K. cowanii A37, and rhizobial strain S. meliloti ARh29 had a positive effect on plant growth indices under salinity
stress. In addition, the single inoculation of non-rhizobial strains without rhizobial strain resulted in an increase
in alfalfa growth indices compared to the plants non-inoculated and the ones inoculated with S. meliloti ARh29
alone under salinity stress, indicating that nodule non-rhizobial strains have PGP potentials and may be a
promising way for improving effectiveness of Rhizobium bio-fertilizers in salt-affected soils.

1. Introduction digestibility. It is widely planted throughout the world, especially in the

Salt stress is one of the major abiotic factors that negatively impacts
crop growth and yield. Salt stress impairs several major processes in
plants, such as photosynthesis, protein synthesis and lipid metabolism,
due to both osmotic effects that result in water deficits and specific ion
effects that can cause toxicity and nutrition imbalance (Munns and
Tester, 2008). Worldwide, approximately 7% of the land on Earth and
20% of the total arable area are affected by salinity (Rizwan et al.,
2015). There is a need to exploit the marginal lands, such as the salt-
affected soil, for forage production. At the present time, leguminous
forage plants have been part of the most critical components in sus-
tainable agriculture and global food security by providing part of the
feed requirement of ruminants for meat and milk production (Barth,
2012; Wang and Brummer, 2012). To meet the requests, leguminous
forage cultivars with high salt tolerance are needed. Alfalfa (Medicago
sativa L.) is a vital perennial leguminous forage crop for its high protein
content, high biomass yield, excellent nutritive value and high
arid and semi-arid areas, for pasture, hay and silage making, and is also
valued highly as a livestock feed directly (Zhang and Wang, 2015). This
forage crop also helps nitrogen (N) incorporation to agricultural sys-
tems, which leads to a reduction in the application of chemical N fer-
tilizers (Fernandez et al., 2017).
It has found that most of commercially available alfalfa cultivars are
deficient in salt tolerance (Liu et al., 2011). Productivity and capacity of
nodule formation and nitrogen-fixation of this plant could be severely
affected by the saline stress as low as of 50 mM NaCl (Bruning and
Rozema, 2013; Liu et al., 2011). In addition, it is known that N2 fixing
legumes are more susceptible to salt stress than N2 non-fixing plants (N-
fertilized plants) (Bouhmouch et al., 2005). Thus, development of al-
falfa cultivars highly resistant to salinity stress is needed. In general,
alfalfa is self-incompatible with inbreeding depression, and insect-as-
sisted pollination. Its cultivars are thus heterogeneous and synthetic.
Therefore, all these traits are difficult to carry out salt tolerance
breeding directly (Zhang and Wang, 2015; Zhu et al., 2005).

⁎
Corresponding authors.
E-mail addresses: hassanetesami@ut.ac.ir (H. Etesami), h.najafi@sanru.ac.ir (H. Najafi Zarini).

https://doi.org/10.1016/j.ecoenv.2018.06.092
Received 17 February 2018; Received in revised form 26 June 2018; Accepted 28 June 2018
Available online 11 July 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
F. Noori et al.
An alternative strategy to improve crop salt tolerance may be to
introduce salt-tolerant bacteria that enhance crop growth under salinity
stress (Etesami and Beattie, 2018; Etesami and Maheshwari, 2018).
Numerous studies have shown that plant growth promoting rhizo-
bacteria (PGPR) have a beneficial impact on plant growth and devel-
opment, especially under stress conditions (Etesami and Maheshwari,
2018). A lot of research has also indicated that plant growth and no-
dulation in legumes are higher in the presence of PGPR (Shaharoona
et al., 2006; Tilak et al., 2006).
It is well known that the rhizobia are not the only nitrogen-fixing
inhabitants of legume nodules (Lu et al., 2017; Martínez-Hidalgo and
Hirsch, 2017). Previously, some endophytic bacteria were isolated from
the nodules of legume plants (Martínez-Hidalgo and Hirsch, 2017). It
has long been known that co-inoculation of rhizobia and endophytic
non-rhizobial bacteria increases the growth and yield of plant, nodu-
lation, and availability of nutrients (i.e., N) (Martínez-Hidalgo and
Hirsch, 2017; Rajendran et al., 2012; Schwartz et al., 2013).
PGPR and endophytic bacteria can increase the plant growth by
various mechanisms (Etesami and Maheshwari, 2018). However, it has
been known that the performance of PGPR/endophytic bacteria is se-
verely affected by environmental factors especially stress factors
(Etesami and Beattie, 2018). For example, Upadhyay et al. (2009)
found that PGPR loose plant growth promoting (PGP) traits with in-
creasing salinity in vitro. Thus, the selection and use of salinity tolerant
PGPR based on both high salt tolerance and efficiency in producing PGP
compounds are potentially of enormous impact in facilitating the
growth of crops in a wide range of saline environments, both natural
salinity and induced salinity. It has been reported that the salinity re-
sistant PGPR obtained from saline environments are more effective at
improving the plant tolerance to salt than those isolated from salt non-
affected habitats (Etesami and Beattie, 2018; Paul and Nair, 2008). So
far, mostly soil (rhizosphere)-isolated microbes have been used for
PGPR/PGPB formulations. However, studies of the nodule microbiome
will better suggest which microbes are effective for developing inocula
(Martínez-Hidalgo and Hirsch, 2017). Although many strategies have
been adopted to improve the stress tolerance in legumes (Medeot et al.,
2010), fewer reports have been published on salinity resistant-PGP
bacteria living in nodules as helper to tolerance to abiotic stress such as
salinity. In addition, few studies of the function of the nodule-associated
non-rhizobial bacteria in nodules have been performed (Martínez-
Hidalgo and Hirsch, 2017).
Keeping in view of the above, the objectives of this study were to: (i)
isolate non-rhizobial and rhizobial bacteria from the root nodules of the
alfalfa grown in saline soils; (ii) characterize these bacterial isolates in
terms of tolerance to salinity and drought and PGP traits; and (iii)
evaluate the effect of effective strains on some physiology and mor-
phology features of alfalfa alone and its symbiosis with Sinorhizobium
(Ensifer) meliloti under salinity stress.
We hypothesized that the root nodules of alfalfa grown in salt-af-
fected soils harbor salinity tolerant PGP bacteria that might improve the
alfalfa salt tolerance by affecting its physiological, morphological and
biochemical processes during stress response.
2. Materials and methods
2.1. Sampling and isolation of nodule endophytic bacteria
To isolate and purify the non-symbiotic and symbiotic bacterial
isolates of alfalfa (Cv, Yazdi) nodules, a number of healthy plants (13
samples) were randomly collected from alfalfa fields (N 34° 43', E 51°
06', and 854 m above sea level) in Qom Province in 2016 during the
growing season (May 15th). Some of physical and chemical traits of
field soil included: soil texture (20% clay, 36% silt, and 44% sand),
loamy; pH, 8.2; electrical conductivity of saturated paste extract (EC),
10.94 dS m−1; organic carbon, 5.7 g kg−1; total nitrogen (Ntot),
5.6 g kg−1; calcium carbonate equivalent (CCE), 89 g kg−1; available
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Ecotoxicology and Environmental Safety 162 (2018) 129–138
potassium (Kavail), 270 mg kg−1; and available phosphorus (Pavail),
7.6 mg kg−1. These traits were measured according to previous
methods (Okalebo et al., 2002). According to a previous standard
procedure (Callow, 1971) and using yeast extract mannitol agar
medium (YMA) supplemented with congo red, the nodule endophytic
bacteria were isolated and purified (Vincent, 1970). To confirm the
surface sterilization process, the aliquots of sterile distilled water used
in the final wash were placed on YMA medium and the plates were
incubated at 28 ± 2 °C for 3–5 days. Bacterial colonies phenotypically
different from each other (e.g., Gram-staining reaction, culture mor-
phology, shape, color, rate of growth, and motility) were selected for
the next experiment. In order to differentiate the symbiotic isolates with
alfalfa from non-symbiotic isolates, the test of nodule formation for
each isolate was checked by inoculation of alfalfa seedlings according
to a previous method (Vincent, 1970). Finally, purified bacterial iso-
lates (fast-growing colonies of bacteria-individual CFU (colony forming
unit)) were selected, sub-cultured onto nutrient agar (NA), maintained
on the respective slants, and stored in a refrigerator at 4 °C for further
studies. In addition, these isolates were also maintained at −80 °C in
nutrient broth (NB) that contained 20% glycerol for long-term storage.
The bacterial isolates were coded (ARh1–ARh30 for rhizobial isolates
and A31–A63 for non-rhizobial isolates).
2.2. Preparation of bacterial inocula
For preparing the bacterial culture, each non-rhizobial bacterial
isolate was grown in 250-ml flasks containing 100 ml NB medium and
shaken for 24 h at 28 ± 2 °C on a rotary shaker at 200 rpm until
(turbid) when logarithmic phase is attained 5 × 108 cells ml−1. For
rhizobial isolates, each isolate was also grown in 250-ml flasks con-
taining 100 ml YMB medium and shaken for 72–120 h at 28 ± 2 °C on
a rotary shaker at 200 rpm until when logarithmic phase is attained
5 × 108 cells ml−1 (Woomer et al., 2011). The cell suspensions were
used in all of the following assays.
2.3. Salinity and drought tolerance assay of nodule endophytic bacteria
To determine the tolerance level to NaCl salinity, each isolate was
assayed by spotting 7 μl of cultures (5 × 108 CFU ml−1) on NA plates
amended with 0, 100, 200, 300, 400, 600, 800, 1000, and 1200 mM
NaCl. After 7 days of incubation at 28 ± 2 °C, the plates were checked
visually for bacterial growth (changes in diameter, state and appear-
ance of colonies) (Khalifa et al., 2016). Since salinity stress results in
osmotic stress, which limits water availability in plants and bacteria,
the tolerance level to drought of these isolates was also assayed (Munns
and Tester, 2008). In order to evaluate the tolerance of the endophytic
isolates (5 × 108 CFU ml−1) to different levels of drought stress, their
growth potential in NB medium containing different concentrations of
polyethylene glycol (PEG)-6000 (0, 202.2, 295.7, 367.7, and 428.4 g
PEG-6000 l−1 NB medium) was assayed. These concentrations are
equivalent to osmotic potential of 0, − 5, − 10, − 15, and − 20 bar,
respectively. After being shaken at 120 rpm on a rotary shaker at
28 ± 2 °C for 72 h, the growth of isolates was determined by mea-
suring the OD of the growth medium at a wavelength of 600 nm (Ali
et al., 2014), which was determined by Absorbance Microplate Readers
(Bio-Tek Elx800, USA). The experiments were done in triplicates.
2.4. Assay of PGP traits of nodule endophytic bacteria
The ability of hydrogen cyanide (HCN) production, the production
of IAA, ACC deaminase activity, phosphate solubilization, and side-
rophore production of all bacterial isolates was determined according
to the methods previously described by Bakker and Schippers (1987),
Gordon and Weber (1951), Penrose and Glick (2003), Sperber (1958),
and Schwyn and Neilands (1987), respectively. The experiments were
done in triplicates. All isolates were also cultured for qualitative
F. Noori et al.
assessment of nitrogen fixation in tubes containing N-free semi-solid
medium (NFb) described by Döbereiner (1989). The presence of a halo
of bacterial growth in the medium was considered as an indicator of
nitrogen fixation. The bacterial growth halo in this medium was com-
pared with a positive control (Azosprillum brasilense, a nitrogen fixing
bacterium).
2.5. Identification of efficient endophytic bacteria
The genomic DNA of the isolates (one rhizobial isolate-ARh29, a
rhizobial isolate with a symbiotic efficiency of more than 100%), and
two non-rhizobial isolates-A36 and A37 grown in NB medium was ex-
tracted using isolation kit (Promega, Madison, WI, USA). The 16S rRNA
gene amplification was performed pursuant to the conditions described
by Edwards et al. (1989) by using general bacterial primers 27F (
5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTA
CGACTT-3′). Sequence of 16S rRNA genes was determined after pur-
ification of PCR products with purification kit (Promega, Madison, WI,
USA) by DNA sequencer by the South Korean Macrogen Corporation
using the 27F Primer. All information about the 16S rRNA gene nu-
cleotide sequence was edited using the Edit sequence (version 5.1)
software. The nucleotide sequences of PCR products were compared
with the nucleotide sequences found in GenBank and the phylogenetic
tree was constructed using the Neighbor Joining method using the
Mega 5 software. The nucleotide sequences identified in this study were
sent to GenBank databases and recorded with various accession num-
bers.
2.6. Effect of efficient strains on alfalfa growth under salinity stress
In this assay, effect of efficient non-rhizobial isolates on some
physiology and morphology features of alfalfa (Cv, Yazdi) was studied
under salinity stress. For this purpose, isolates A36 and A37 of non-
rhizobial endophytic bacteria and isolate ARh29 as rhizobial isolate
were selected and tested in this assay. This assay was performed as a
completely randomized design (CRD) with factorial arrangement in
three replications in potted soil (2 kg). The research was carried out in a
research greenhouse located at Department of Soil Science, University
of Tehran, Iran. The soil (a light textured soil) used in pots was a poor
soil in terms of bacteria and micro-and macronutrients. The experi-
mental treatments included five inoculation conditions, (i) the seedlings
co-inoculated with non-rhizobial endophytic bacteria A36 and A37; (ii)
the seedlings inoculated with rhizobial bacterium ARh29; (iii) the
seedlings co-inoculated with isolates A36, A37, and ARh29; (iv) the
seedlings non-inoculated with bacterial isolates (negative control); and
(v) the seedlings non-inoculated with bacterial isolates and fertilized
with N (positive control), as the first factor, and five salinity levels, (i)
the alfalfa plants grown at 0 mM NaCl; (ii) the alfalfa plants grown at
50 mM NaCl; (iii) the alfalfa plants grown at 100 mM NaCl; (iv) the
alfalfa plants grown at 150 mM NaCl; and (v) the alfalfa plants grown at
200 mM NaCl, as the second factor. Since these bacterial isolates were
co-inoculated on alfalfa, the antagonistic assay was initially performed
on these isolates as described by Etesami et al. (2014). None of the
isolates had a negative effect on the growth of each other. In order to
sow alfalfa, plastic pots (diameter, 15 cm and height, 14 cm) without
drainage were used. Each pot was disinfected with sodium hypochlorite
5% solution and washed well with sterilized distilled water. To each
pot, 2 kg of non-sterile soil passed through a 4 mm sieve was added.
Depending on the experimental treatments, different saline solutions
were prepared using NaCl to create different salinity conditions. In each
pot, after being surface-sterilized (respectively for 7 s with 96% alcohol,
60 s with 5% sodium hypochlorite and 10 times washing with sterile
distilled water), the 20 germinated healthy seeds of alfalfa (Cv, Yazdi),
obtained from Seed and Plant Improvement Institute, were planted in a
1 cm depth of soil surface. After 14 days of sowing, each seedling was
inoculated with one ml of bacterial suspension according to the relevant
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Ecotoxicology and Environmental Safety 162 (2018) 129–138
treatment. After 32 days of sowing (18 days after inoculation), salinity
treatments were applied. To avoid salt shock, NaCl solutions were ap-
plied to pots in increments of 50 mM every 48 h, until final salt con-
centrations of 100, 150, and 200 mM were reached. The amount of
20 kg N per hectare of urea fertilizer was added to all pots as a starter.
Nutritional need of the plants was provided by nitrogen-free Hoagland
solution. Hoagland solution containing N was added to plants con-
sidered as positive controls. Soil moisture content was maintained by
weight basis and daily at 80% of the field capacity. During the ex-
periment, irrigation with sterile distilled water was performed. All pots
were maintained in greenhouse with the light intensity of 15000 lx, at
25 ± 2 °C and light period of 12 h for 70 days.
2.6.1. Measurements
After the end of the growth period, alfalfa plants (five plants ran-
domly from each pot) were cut off from the soil surface of the pots.
Samples (shoot) were washed with distilled water, dried at 70 °C until
they reached constant weight, and weighed and then milled. Also, the
roots were removed from soil and placed in an oven for 72 h at 70 °C in
order to stabilize their final dry weight and then the root dry weight
was measured. Using an electric mill, these samples were then pow-
dered. The total N content of the shoot was measured by the titration
method after distillation using the Kjeldahl method (Okalebo et al.,
2002). The digestion of the plant aerial parts was done by dry ashing
method. In order to prepare the extracts necessary for the measurement
of K, Na, and P, after the addition of 1N HCl, the extracts were filtered
and analyzed by flame photometer (ELE) and spectrophotometer (Shi-
madzu UV3100), respectively, according to previous methods (Okalebo
et al., 2002).
For measuring proline content, a 0.5 g frozen sample was crushed in
a mortar with liquid N and then extracted with 5 ml of 3% (w/v) sul-
phosalycylic acid. After the homogenate was centrifuged at 18,000 × g
for 10 min, 2 ml of the supernatant was transferred to a test tube.
Afterward, 2 ml glacial acetic acid and 3 ml ninhydrin reagent were
added to the test tube and kept for 40 min at 100 °C. Finally, toluene
was added to the mixture reaction for extracting the product. After
shaking, the tube was left for 15 min. The mixture gradually became
two-phase and the upper phase absorbance was measured at 520 nm
against toluene (Bates et al., 1973). The proline content was determined
from calibration curve using L-proline as standard and expressed as
µg g−1 leaf fresh weight (FW).
Among antioxidant enzymes, activity of catalase (CAT) and super-
oxide dismutase (SOD) was measured in this study. Concisely, the
samples of fresh leaf were homogenized with a mortar in 100 mM
phosphate buffer (pH 6.8) containing 0.1 mM EDTA and 1% PVP
(polyvinylpyrrolidone). After the homogenate was centrifuged at
15000 × g for 15 min, the supernatant was utilized as the source of
enzymes. All the operations were carried out at 4 °C. For the activity of
SOD, the reaction combination included 50 mM sodium carbonate (pH
10.2), 12 mM L-methionine, 50 mM HEPES-KOH buffer (pH 7.8) in-
cluding 0.1 mM EDTA, 75 µM nitroblue tetrazolium (NBT), and 1 µM
riboflavin. The activity of SOD was determined based on its ability to
prevent the reduction of NBT by superoxide anion produced by the
system of riboflavin below 4000 W (light intensity) at 25 °C
(Giannopolitis and Ries, 1977). An SOD activity unit is considered an
enzyme value that results in 50% inhibition of the reduction rate of
nitroblue tetrazolium (NBT) at 560 nm. The reaction mixture for the
CAT activity was contained 100 mM phosphate buffer (pH 7) and
15 mM H2O2. The decomposition of H2O2 (hydrogen peroxide) was
absorbed at 240 nm for 3 min (ε = 39.4 mM cm−1) (Aebi, 1984). As
well as, the soluble protein was calculated according to Bradford (1976)
by using bovine serum albumin (BSA) as standard. The specific enzyme
activity for these enzymes was expressed as unit mg−1 protein.
F. Noori et al. Ecotoxicology and Environmental Safety 162 (2018) 129–138

Fig. 1. A heatmap diagram of characteristics of salinity and drought tolerance of rhizobial isolates (ARh1-ARh30) and non-rhizobial isolates (A31–A63) isolated from
root nodules of alfalfa grown in salt-affected-soils. The color bar on top shows the relative abundance of isolates tolerant to salinity and drought stress. Regarding the
color scheme, red indicates higher tolerance of bacterial isolates to salinity and drought stress and blue indicates lower tolerance of bacterial isolates to salinity and
drought stress or sensitivity of the bacterial isolates to salinity and drought stress. A, drought stress (osmotic potential of 0 bar); B, drought stress (osmotic potential of
-5 bar); C, drought stress (osmotic potential of -10 bar); D, drought stress (osmotic potential of -15 bar); E, drought stress (osmotic potential of -20 bar); F, salinity
stress (400 Mm NaCl); and G, salinity stress (1200 Mm NaCl) (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.).

Fig. 2. A heatmap diagram of characteristics of promoting plant growth of rhizobial isolates (ARh1-ARh30) and non-rhizobial isolates (A31–A63) isolated from root
nodules of alfalfa grown in salt affected-soils. The color bar on top shows the relative abundance of isolates having PGP traits. Regarding the color scheme, higher
values indicate higher ability of bacterial isolates to produce plant growth promoting (PGP) traits and lower values indicate lower ability of bacterial isolates to
produce PGP traits or without ability to produce PGP traits. A, IAA production; B, organic P solubilization; C, inorganic P solubilization; D, N2 fixation; E, ACC
deaminase activity; and F, HCN production.

132
F. Noori et al.
2.7. Statistical analysis
Analysis of variance (ANOVA) of data and statistical calculations
were performed using MSTAT and SAS computer programs (SAS
Institute, Cary, NC, USA). Charts were plotted using prism 6 software.
Comparison of mean of data was done by Duncan's multiple-range tests
at 5% level. The data were reported as means ± the standard devia-
tions.
3. Results
3.1. Isolation of nodule endophytic bacteria and their characterizations
A total of 63 distinct bacterial isolates from alfalfa surface-sterilized
nodules were isolated. Thirty out of 63 isolates were rhizobial isolates
that were able to form nodules on alfalfa roots (ARh1-ARh30). The
ability of all 63 isolates to tolerate salinity and drought was evaluated
(Fig. 1). All isolates were able to grow at salt concentrations up to
400 mM NaCl, while only four isolates (A36, A37, A38, and A44) could
grow at salt concentrations up to 1200 mM NaCl. Most of the bacterial
isolates were partially capable of growing at an osmotic potential to
− 5 bar, but only four non-rhizobial isolates were able to tolerate os-
motic potential to − 20 bar (Fig. 1). The non-rhizobial isolates A36,
A37, A38, and A44 showed similar responses to salinity and drought
stress and were more tolerant than other isolates. The ability to tolerate
salinity of isolates using sodium chloride salt showed that with in-
creasing salt concentration, the growth of bacteria decreased. In addi-
tion, with increasing concentration of PEG-6000 in culture media, op-
tical density (OD600) showed a decreasing trend as a criterion for
bacterial growth. Among 63 bacterial isolates, non-rhizobial isolates
A36, A37, A38, and A44 showed the highest drought and salinity tol-
erance (Fig. 1).
The potential of 63 isolates was also evaluated for the character-
istics of promoting plant growth. The ability to produce PGP traits
varied among isolates (Fig. 2). As shown in Fig. 2, all isolates were
positive for IAA production. The amount of the IAA produced by these
isolates varied from 2 to 63 µg ml−1, with the highest production
amount of this hormone among non-rhizobial isolates. Compared to the
ability of the isolates to inorganic P solubilization, these bacterial iso-
lates showed greater potential for solubilization of organic P. None of
the rhizobial and non-rhizobial isolates were able to produce side-
rophore. All rhizobial isolates obtained from root nodules were able to
grow in an N-free culture medium, while only eight non-rhizobial iso-
lates were able to grow at this culture medium. None of the rhizobial
isolates were able to use ACC as the only source of N, while only non-
rhizobial isolates A36, A37, A38, and A44 were positive for the pro-
duction of the enzyme ACC deaminase. Except for some non-rhizobial
and rhizobial isolates, the isolates were not positive for HCN produc-
tion.
Table 1
Characteristics of promoting plant growth and salinity and drought tolerance of effective non-rhizobial isolates A36 and A37 and rhizobial isolate ARh29 isolated
from root nodules of alfalfa selected in this study.
Strain IAA production Inorganic P Organic P Siderophore ACC
solubilization solubilization production deaminase
(µg ml−1)
ARh29 7.50 + – – –
A36 62.16 + + – +
A37 35.16 + + – +
+ , positive activity; -, negative activity.
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Ecotoxicology and Environmental Safety 162 (2018) 129–138
3.2. Identification of effective endophytic isolates
Based on their tolerance to salinity and drought and having the
highest potential in terms of PGP characteristics (Table 1), two non-
rhizobial isolates A36 and A37 were selected and identified. Among
rhizobial isolates, rhizobial isolate ARh29 was identified. This rhizobial
isolate indicated a symbiotic efficiency of more than 100% (data not
shown). For finding sequences similar to the 16S rRNA gene sequence
of these isolates, databases in the GeneBank were checked. Identifica-
tion of the 16S rRNA gene of the isolates A36, A37, and ARh29 was
shown to be closely related to Klebsiella sp., Kosakonia cowanii, and
Sinorhizobium meliloti, respectively. The 16S rRNA gene sequences of
A36, A37, and ARh29 were closely similar to strains Klebsiella sp. M5al
(accession number CP020657), K. cowanii Esp_Z (accession number
CP022690), and S. meliloti B399 (accession number CP019488) on the
BLAST search in the NCBI, respectively. The corresponding dendrogram
was drawn using the sequences of the strains and representative se-
quences from the databases (Fig. 3). The nucleotide sequences assigned
to this study were sent to the GeneBank database and registered with
the accession numbers MH201369, MH201368, and MG586242 for
strains A36, A37, and ARh29, respectively.
3.3. Effect of effective bacterial strains on shoot N concentration and root
dry weight
Analysis of variance of the interaction effect between salinity levels
and bacterial treatment on N concentration and root dry weight was not
significant in this study (ANOVA table not shown). However, the
average N concentration of the shoot and the average root dry weight
decreased significantly with increasing salinity level (Fig. 4A and C). In
this study, the use of plant growth promoting bacteria (both rhizobial
strains and non-rhizobial ones) significantly increased N concentration
and root dry weight and the highest amount of shoot N and root dry
weight was observed in plants co-inoculated with non-rhizobial strains
Klebsiella sp. A36 and K. cowanii A37 and rhizobial strain S. meliloti
ARh29 (a 31.54% increase and a 14.92% increase in N concentration
compared to negative and positive controls, respectively), while plants
co-inoculated with non-rhizobial strains A36 and A37 and the plants
inoculated with S. meliloti ARh29 were in the next ranks in terms of N
content and root dry weight compared with positive and negative
controls, respectively (Fig. 4B and D). An interesting result of this as-
sessment was that the plants inoculated with non-rhizobial strains
(A36 + A37) were able to take up the amount of N statistically
equivalent to the plants co-inoculated with non-rhizobial strains and
rhizobial strain (A36 + A37 + ARh29). This indicates that non-rhizo-
bial bacteria had a considerable role in the fixation of N2 in the absence
of S. meliloti ARh29. On roots of alfalfa plants inoculated with non-
rhizobial strains, no nodule was observed, indicating that the soil used
in the pots did not contain any indigenous rhizobial bacteria symbiotic
with the alfalfa.
HCN N2 fixation Tolerance to salinity Tolerance to
production drought
400 mM 1200 mM (Osmotic
NaCl NaCl potential of
− 20 bar)
– + + – –
+ + + + +
+ + + + +
F. Noori et al. Ecotoxicology and Environmental Safety 162 (2018) 129–138

Fig. 3. Phylogenetic tree constructed using 16S rRNA gene

sequences, available in the GenBank database, employing
the Neighbor-joining method. Bootstrap values based on
1000 replications were listed as percentages at the nodes.
The scale bar indicates genetic distance. The GenBank
accession number is given in parentheses for each or-
ganism. The bacterium Cuniculiplasma divulgatum was
considered as outgroup.

3.4. Effect of superior bacterial strains on shoot dry weight in this study (Table 2). At all levels of salinity, bacterial strains could
augment the dry weight of the plant compared with non-inoculated
Analysis of variance of the interaction effect between salinity levels control. However, the highest dry weight of shoot was recorded in
and bacterial treatment on shoot dry weight was significant (p < 0.01) plants co-inoculated with S. meliloti ARh29 and non-rhizobial strains

Fig. 4. Effect of non-rhizobial strains and rhizobial strain on the N content of shoot and root dry weight of alfalfa plant grown under salinity stress in greenhouse
conditions for 70 days. Means ± SD with different letters are significantly different at the 5% level according to Duncan's multiple-range tests. A36, Klebsiella sp.;
A37, K. cowanii; and ARh29, S. meliloti.

134
 F. Noori et al.

Table 2
Interaction effect of non-rhizobial strains Klebsiella sp. A36 and K. cowanii A37 and rhizobial strain S. meliloti ARh29 on some characteristics of physiology and morphology of alfalfa plant grown under salinity stress in
greenhouse conditions for 70 days. Means ± SD with different letters are significantly different at the 5% level according to Duncan's multiple-range tests.
Treatments Shoot DW Na+ K+ Leaf K+/Na+ Shoot P content (%) Proline SOD activity CAT activity
(mg plant−1) (mg g−1 leaf DW) (mg g−1 leaf DW) (µg g−1 leaf FW) (unit mg−1 protein) (unit mg−1 protein)

0 mM NaCl Strains A36 + A37 138.9 ± 3.2 bc 2.46o ± 0.22 p 53.7 ± 0.79 a 21.97 ± 1.72 c 0.76 ± 0.02 b 3.13 ± 0.21 mno 12.7 ± 0.29 kl 1.63 ± 0.14 kl
Strain ARh29 142.40 ± 5.8 b 1.31 ± 0.25 q 52.3 ± 0.88 b 41.20 ± 7.02 a 0.53 ± 0.01 e 2.67 ± 0.21 n-q 11.6 ± 0.31 m 1.44 ± 0.07 kl
Strains A36 + A37 + ARh29 152.20 ± 2.9 a 1.83 ± 0.33 pq 52.3 ± 0.41 b 29.60 ± 5.75 b 0.87 ± 0.02 a 3.40 ± 0.22 lmn 13.6 ± 0.42 j 1.82 ± 0.09 kl
No bacteria 89.80 ± 2.5 lm 2.71 ± 0.20 o 47.0 ± 1.00 fg 17.40 ± 1.04 d 0.25 ± 0.02 jk 1.94 ± 0.12 q 8.20 ± 0.15 p 0.84 ± 0.07 m
No bacteria + Nitrogen 127.80 ± 2.6 ef 1.96 ± 0.16 pq 49.3 ± 0.78 cd 25.40 ± 2.25 c 0.30 ± 0.01 i 2.27 ± 0.21 pq 9.9 ± 0.48 o 1.51 ± 0.12 kl
50 mM NaCl Strains A36 + A37 132.20 ± 1.7 de 8.50 ± 0.26 m 49.9 ± 0.43 c 5.87 ± 0.19 efg 0.56 ± 0.01 e 3.93 ± 0.21 klm 13.7 ± 0.37 ij 2.37 ± 0.21 j
Strain ARh29 135.10 ± 1.0 cd 7.45 ± 0.05 n 48.5 ± 1.04 de 6.50 ± 0.12 ef 0.44 ± 0.02 g 3.83 ± 0.21 klm 13.1 ± 0.29 jk 2.40 ± 0.08 j
Strains A36 + A37 + ARh29 136.90 ± 1.2 cd 7.05 ± 0.21 n 50.0 ± 0.61 c 7.11 ± 0.29 e 0.70 ± 0.02 c 4.13 ± 0.33 kl 15.7 ± 0.24 h 3.17 ± 0.17 gh
No bacteria 80.40 ± 0.8 n 10.20 ± 0.21 l 42.3 ± 0.49 k 4.17 ± 0.10 e-h 0.19 ± 0.01 mn 2.37 ± 0.12 opq 9.60 ± 0.29 o 1.30 ± 0.22 lm
No bacteria + Nitrogen 124.90 ± 2.3 f 9.55 ± 0.40 l 44.7 ± 0.30 j 4.69 ± 0.17 e-h 0.20 ± 0.0 lm 2.93 ± 0.12 nop 12.3 ± 0.29 l 1.86 ± 0.13 k
100 mM NaCl Strains A36 + A37 123.80 ± 1.8 fg 14.70 ± 0.31 j 47.6 ± 0.71 ef 3.24 ± 0.12 e-h 0.44 ± 0.02 g 7.13 ± 0.25 ij 17.7 ± 0.30 g 3.47 ± 021 g
Strain ARh29 119.10 ± 1.3 g 13.40 ± 0.30 k 47.1 ± 0.73 fg 3.52 ± 0.13 e-h 0.36 ± 0.01 h 7.27 ± 0.58 i 16.1 ± 0.25 h 3.47 ± 0.25 g

135
Strains A36 + A37 + ARh29 124.70 ± 1.0 f 14.40 ± 0.31 j 48.6 ± 0.27 cde 3.37 ± 0.09 e-h 0.60 ± 0.01 d 8.43 ± 0.46 h 19.7 ± 0.29 f 4.23 ± 0.29 f
No bacteria 72.97 ± 2.3 o 19.00 ± 0.42 g 38.9 ± 0.78 m 2.05 ± 0.07 fgh 0.13 ± 0.01 o 4.53 ± 0.17 k 10.8 ± 0.33 n 1.63 ± 0.17 kl
No bacteria + Nitrogen 107.70 ± 1.6 h 17.10 ± 0.50 h 42.2 ± 1.18 k 2.47 ± 0.13 fgh 0.18 ± 0.01 n 6.37 ± 0.25 j 14.4 ± 0.34 i 2.96 ± 0.24 ghi
150 mM NaCl Strains A36 + A37 101.40 ± 2.1 ij 17.70 ± 0.42 h 46.1 ± 0.22 ghi 2.61 ± 0.07 e-h 0.36 ± 0.01 h 12.60 ± 0.74 e 24.1 ± 0.25 d 4.86 ± 0.18 de
Strain ARh29 100.40 ± 2.4 ij 16.20 ± 0.18 i 46.4 ± 0.30 fgh 2.87 ± 0.02 e-h 0.31 ± 0.00 i 11.30 ± 0.60 f 23.2 ± 0.60 e 4.78 ± 0.13 e
Strains A36 + A37 + ARh29 104.20 ± 2.2 hi 16.20 ± 0.35 i 46.7 ± 0.56 fg 2.89 ± 0.04 e-h 0.47 ± 0.01 f 14.30 ± 0.33 d 26.8 ± 0.27 c 6.07 ± 0.21 c
No bacteria 65.93 ± 3.3 p 23.30 ± 0.38 c 36.2 ± 0.20 n 1.55 ± 0.02 gh 0.10 ± 0.00 po 7.03 ± 0.21 ij 13.8 ± 0.50 ij 2.53 ± 0.21 ij
No bacteria + Nitrogen 86.77 ± 3.3 m 20.30 ± 0.25 f 40.8 ± 0.46 l 2.01 ± 0.02 fgh 0.14 ± 0.01 o 9.64 ± 0.45 g 18.2 ± 0.21 g 4.20 ± 0.41 f
200 mM NaCl Strains A36 + A37 94.37 ± 2.1 kl 22.10 ± 0.20 d 44.0 ± 0.35 j 1.99 ± 0.01 fgh 0.26 ± 0.01 j 18.20 ± 0.69 b 27.6 ± 0.29 b 7.27 ± 0.33 b
Strain ARh29 90.37 l ± 1.6 m 20.0 ± 0.42 f 45.0 ± 0.53 ij 2.25 ± 0.06 fgh 0.23 ± 0.01 kl 17.30 ± 0.26 c 26.4 ± 0.37 c 6.83 ± 0.57b
Strains A36 + A37 + ARh29 97.27 ± 2.3 jk 21.20 ± 0.27 e 45.2 ± 9.26 hij 2.14 ± 0.04 fgh 0.32 ± 0.01 i 19.70 ± 0.44 a 30.6 ± 0.58 a 8.57 ± 0.30 a
No bacteria 61.33 ± 2.5 p 27.70 ± 0.48 a 33.1 ± 0.66 o 1.20 ± 0.04 h 0.09 ± 0.00 q 9.20 ± 0.45 g 17.7 ± 0.27 g 2.92 ± 0.09 hi
No bacteria + Nitrogen 74.87 ± 2.9 o 24.90 ± 0.08 b 38.3 ± 0.55 m 1.53 ± 0.02 gh 0.13 ± 0.00 op 13.90 ± 0.25 d 22.8 ± 0.29 e 5.30 ± 0.43 d
Source of variation Shoot DW Na+ K+ K+/Na+ P Proline SOD activity CAT activity
Salinity levels (S) ** ** ** ** ** ** ** **
Bacterial strains (B) ** ** ** ** ** ** ** **
S × B (Interaction) ** ** ** ** ** ** ** **

A36, Klebsiella sp.; A37, Kosakonia cowanii; ARh29, Sinorhizobium meliloti; DW, dry wright; FW, fresh weight; CAT, activity of catalase; SOD, activity of superoxide dismutase; and **, Significant at p < 0.01.
Ecotoxicology and Environmental Safety 162 (2018) 129–138
F. Noori et al.
(A36 + A37 + ARh29) at all salinity levels. In addition, there were not
any symptoms of a disease or other specific signs on plants inoculated
with these strains.
3.5. Effect of effective bacterial strains on Na+ and K+ concentration in
leaves
The results of mean comparison showed that the highest amount of
Na+ and K+ was observed in the negative control (No bacteria + 200
mM NaCl) and in the plants inoculated with non-rhizobial strains
(A36 + A37) and without salinity (0 mM NaCl), respectively (Table 2).
Compared to non-inoculated plants, inoculated plants absorbed the
lower amount of Na+. An increase in Na+ concentration in inoculated
and non-inoculated plants can also be due to the abundance of Na+ ions
in the root environment and the sodium ion toxicity-mediated re-
tardation of plant growth in higher salinity (contrary to dilution effect).
At all salinity levels, bacterial strains increased K+ uptake in plants, but
the highest K+ absorption was observed in plants co-inoculated with
Klebsiella sp. A36, K. cowanii A37, and rhizobial strain S. meliloti ARh29
(A36 + A37 + ARh29), followed by plants inoculated with non-rhi-
zobial strains Klebsiella sp. A36 and K. cowanii A37 (A36 + A37). Al-
though K+ concentration increased under salt stress, this increase was
negligible as compared to Na+ concentration. The highest ratio of K+/
Na+ was observed in non-stressed plants with S. meliloti ARh29. As the
salinity levels increased, the K+/Na+ ratio decreased in inoculated and
non-inoculated plants.
3.6. Effect of effective bacterial strains on P concentration in shoot
The average concentration of P decreased significantly with in-
creasing salinity level. The results of mean comparison showed that the
effect of bacterial treatments on P concentration was significantly
higher than the control (plants non-inoculated with bacterial strains),
and its highest concentration was observed in the plants co-inoculated
with rhizobial strain and non-rhizobial strains (A36 + A37 + ARh29)
and without salinity stress (Table 2). In addition, in the higher salinity
levels (50, 100, 150, and 200 mM NaCl), the highest concentration of P
was also observed in bacterial treatment of A36 + A37 + ARh29, and
treatment of A36 + A37 was in the next rank.
3.7. Effect of effective bacterial strains on proline concentration and
antioxidant enzymes
Analysis of variance of data showed that the effect of salinity stress
and bacterial levels on proline concentration and antioxidant enzymes
was significant (p < 0.01) (Table 2). Activity of antioxidant enzymes
augmented with enhancing salinity stress initially and indicated a di-
minishing trend in high salinity. At all salinity levels, plants inoculated
with bacterial strains had higher proline concentration and enzymatic
activity (CAT and SOD) compared with non-inoculated plants. Com-
pared to low salinity, proline concentration and activity of the enzymes
CAT and SOD were higher in plants inoculated and grown under high
salt concentrations. The highest concentration of proline (19.70 µg g 1̶
leaf FW) and activity of enzymes of CAT (8.60 unit mg−1 protein) and
SOD (30.60 unit mg−1 protein) were observed in plants co-inoculated
with Klebsiella sp. A36, K. cowanii A37, and rhizobial strain S. meliloti
ARh29 (A36 + A37 + ARh29) and subsequently in plants inoculated
with non-rhizobial strains of Klebsiella sp. A36 and K. cowanii A37
(A36 + A37), compared to controls (no bacteria and no bacteria +
nitrogen).
4. Discussion
It is well known that the bacteria isolated from saline environments
are more efficient to improve the plant tolerance to salt compared to the
bacteria isolated from non-saline environments (Etesami and Beattie,
136
Ecotoxicology and Environmental Safety 162 (2018) 129–138
2018). It has also been found that the rhizobia are not the only ni-
trogen-fixing inhabitants of legume nodules (Lu et al., 2017; Martínez-
Hidalgo and Hirsch, 2017). In the present study, we isolated a set of
salinity-tolerant rhizobial and non-rhizobial endophytic bacteria from
root nodules of alfalfa grown in saline soils (EC, 10.94 dS cm−1). Iso-
lation of non-rhizobial bacteria from root nodules of alfalfa (Lai et al.,
2015) and other legumes (Korir et al., 2017; Leite et al., 2017) has also
been reported in previous studies. The tolerance of these isolates to
salinity and drought was different. According to previous findings,
these differences may be related to the status of ion pumps, including
sodium pumps, and the regulation of ions by different bacteria (Preiss
et al., 2015). In addition to their tolerance to salinity and drought, these
endophytic isolates were also able to produce PGP characteristics. In
this study, all isolates were positive for IAA production. Dilfuza (2011)
reported that 90% of the bacteria associated with plants were able to
produce the IAA hormone. It is known that bacterial metabolite of IAA
is able to elevate protection of bacterial cells against different abiotic
stresses such as high salt concentration (Bianco et al., 2006). The ability
to produce PGP properties like N2 fixing activity, IAA, ACC deaminase,
and phosphate solubilization in endophytic bacteria has been reported
in previous studies (Bulgarelli et al., 2013; Etesami and Alikhani, 2016).
In this study, the best non-rhizobial isolates and rhizobial isolate were
identified, which were similar to Klebsiella sp., K. cowanii, and S. meli-
loti, respectively. To our knowledge, this is the first study showing
Klebsiella sp. and K. cowanii as salinity and drought endophytic bacteria
isolated from alfalfa root nodules with multiple PGP traits (Table 1). In
previous studies, Chimwamurombe et al. (2016) and Almeida Lopes
et al. (2016) also isolated endophytic strains Kosakonia cowanii from
bean and soybean plant. The strain A37 identified in this study was
positive for production of IAA and ACC deaminase and phosphate so-
lubilization. The ability to fix nitrogen in this genus has also been re-
ported (Chen et al., 2014). Martínez-Hidalgo and Hirsch (2017) also
stated that some nodule non-rhizobial bacteria can fix nitrogen. Kleb-
siella sp., as an endophytic bacterium, has also been isolated from
Glycine and Vicia (Martínez-Hidalgo and Hirsch, 2017). This bacterium
has increased plant growth by fixing nitrogen, IAA production, and
phosphate solubilization (Martínez-Hidalgo and Hirsch, 2017).
Co-inoculation of alfalfa with Klebsiella sp. A36, K. cowanii A37, and
S. meliloti ARh29 increased the growth indices of the alfalfa plant at all
levels of salinity compared with the inoculation of these isolates alone.
Non-rhizobial strains Klebsiella sp. A36 and K. cowanii A37 significantly
also increased plant growth in the absence of rhizobial strain S. meliloti
ARh29. These bacteria could even provide plant N in the absence of S.
meliloti ARh29 and nitrogen (Fig. 4B), which was an interesting result of
this research. Stajković et al. (2009) also isolated non-rhizobial en-
dophytic strains (i.e., Brevibacillus chosinensis and Microbacterium tri-
chothecenolyticum) from alfalfa nodules that were able to stimulate al-
falfa growth in the presence and absence of rhizobial strain S. meliloti.
Further increase in the growth of alfalfa plants inoculated with non-
rhizobial strains of Klebsiella sp. A36 and K. cowanii A37compared to
that of the alfalfa plants inoculated with rhizobial strain S. meliloti
ARh29 may be attributed to the higher tolerance of the non-rhizobial
strains (A36 and A37) to salinity and drought and ACC deaminase
production capacity of these strains as well as to their higher ability to
produce IAA hormone (Table 1). In previous studies, Klebsiella sp. could
also confer enhanced tolerance to salinity and plant growth promotion
in oat seedlings (Avena sativa L.) (Sapre et al., 2018) and in wheat
(Triticum aestivum L.) (Singh et al., 2015).
Under salinity stress, absorption of nutrients is decreased due to
reduced root growth caused by stress ethylene production (Siddikee
et al., 2011) and reduction of the activity of ion transporter by sodium
(Yao et al., 2010). It has been proven that the bacteria capable of
producing ACC-deaminase enzyme (by decreasing stress ethylene) and
IAA can increase the growth of plant cells and/or plant tissue elonga-
tion (increased root growth and root length), which increases greater
root surface area and enables the plant to get more nutrients (i.e., P and
F. Noori et al.
K) and water from the soil (Etesami and Maheshwari, 2018). According
to this finding, the alfalfa plants inoculated with these non-rhizobial
strains (A36 + A37) showed a better rooting system compared with the
plants non-inoculated with these strains (Fig. 4D).
The generation of reactive oxygen species (ROS) is one of the ad-
verse effects of salinity. It has been reported that plant-associated
bacteria can increase the plant tolerance by improving the activity of
antioxidant enzymes and osmotic adjustment capacity (Etesami and
Maheshwari, 2018; Grover et al., 2011; Shrivastava and Kumar, 2015).
In the present study, proline concentration and activity of SOD and CAT
were significantly increased by co-inoculation of Klebsiella sp. A36 + K.
cowanii A37 + S. meliloti ARh29 at all salinity levels compared to
control. It was found that Rhizobium symbiosis also increased alfalfa salt
tolerance by improving the activity of antioxidant enzymes and osmotic
adjustment capacity (Wang et al., 2016).
Studies indicate that salinity reduces the uptake/accumulation of
plant nutrients (e.g., N, P, and K) and the translocation of water in
stressed plants (Etesami and Maheshwari, 2018). It is known that
salinity-tolerant-PGPR are able to enhance the uptake of K+ ion by
mediating the expression of an ion high-affinity K+ transporter
(AtHKT1) in plant under saline conditions and, in turn, a higher K+/
Na+ ratio that favors salinity tolerance (Etesami and Beattie, 2018;
Rojas-Tapias et al., 2012). In this study, the highest content of P and K
was observed in the plant inoculated with non-rhizobial bacteria
(Table 2), which can probably be due to synergistic effects of IAA and
ACC deaminase on root length, and finally P and K uptake of soil by
root and their transfer to plant aerial parts.
In most previous studies (Elkoca et al., 2010; Figueiredo et al., 2008;
Korir et al., 2017; Medeot et al., 2010; Tsigie et al., 2011), it has been
reported that the efficiency of N2 fixation and the growth indices of
legume plants by rhizobial bacteria increase in the presence of PGPR.
For the first time in this study, we showed that N2 fixing-non rhizobial
bacteria (Klebsiella sp. A36 + K. cowanii A37) could provide plant N and
increase plant growth indices without rhizobial bacteria and nitrogen
(Fig. 4B). This indicates that the non-rhizobial bacteria identified in this
study were able to fix N2. According to the results obtained from this
study, we confirmed the hypothesis that salinity tolerant bacteria, iso-
lated from the root nodules of alfalfa grown in saline soils, can have a
significant role in improving plant growth and alfalfa tolerance to
salinity stress in the presence and absence of rhizobial bacterium
symbiotic to alfalfa plant (S. meliloti).
5. Conclusions
The results of this study clearly indicate that the root nodules of
alfalfa plants grown in saline areas can be a useful source of drought
and salinity tolerant bacteria with multiple PGP traits. Although many
of these nodule non-rhizobial bacteria were not capable of fixing ni-
trogen, they had the potential to increased alfalfa growth under salinity
conditions both in the presence and in absence of rhizobial bacterium
symbiotic to alfalfa plant (S. meliloti). This knowledge will be useful in
defining strategies to apply these bacteria as bio-inoculants by them-
selves or combined with rhizobial bacteria. Such an approach will en-
hance rhizobial performance or persistence as well as diminish the
usage of chemical fertilizers. According to the findings of this study,
PGP bacteria of Klebsiella sp. A36 and K. cowanii A37 may be suitable
candidates as tolerant bacteria in different degrees to salinity and can
be used in the formulation of agricultural products (as a cheaper al-
ternative to farmers in production of alfalfa in saline soils) after being
tested under field conditions.
Acknowledgments
We wish to thank University of Tehran and Sari Agricultural
Sciences and Natural Resources University for providing the necessary
facilities for this study.
137
Ecotoxicology and Environmental Safety 162 (2018) 129–138
Conflict of interest
The authors declare that they have no conflict of interest.
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